Restriction endonucleases are essential tools in genetic engineering, cloning and DNA diagnostics. N.BstNBI is a natural occurring nicking endonuclease which only breaks one strand of double-stranded DNA, and it is the only nicking enzyme that is commercially available. The proposed research is about engineering Type IIs endonucleases into nicking enzymes. In Phase I research, we have cloned and characterized the nicking endonuclease N.BstNBI and two related type IIs endonucleases, PleI and MlyI. Our results showed that type IIs endonucleases PleI and MlyI carry out double-stranded cleavage in a two-step process, in which the DNA is first nicked and then further cleaved on second strand by another enzyme molecule via dimerization. In the case of N.BstNBI, it is the dimerization function that was probably affected by mutation, resulting in a unique endonuclease which nicks DNA. We have successfully converted MlyI into a nicking endonuclease by disrupting its dimerization function. Thus, we have demonstrated that it is possible to convert Type IIs endonucleases into nicking enzymes by mutations. We will apply the techniques used in Phase I as well as additional strategies to convert more Type IIs endonucleases into new nicking enzymes. PROPOSED COMMERCIAL APPLICATIONS: Potentially can lead to the conversion of type IIs restriction endonucleases into nicking enzymes which have great applications in DNA amplification technology (SDA, RCA) and research (DNA replication, repair, etc.).